Journal: Frontiers in Genetics

Article Title: Exosomes containing long non-coding RNA AGAP2-AS1 promote the differentiation of CD4 + T cells through the miR-424-5p/SGK1 axis in psoriasis

doi: 10.3389/fgene.2025.1521470

Figure Lengend Snippet: ASAP2-AS1 promotes the differentiation of Th1 and Th17 in CD4 + T cells and serves as a sponge for miR-424-5p. (A) Fluorescence microscopy was used to detect the subcellular localization of AGAP2-AS1 in CD4 + T cells. AGAP2-AS1 was labeled with Cy3, cell nuclei were counterstained with DAPI (blue). (B) The expression of AGAP2-AS1 in CD4 + T cells after co-incubation of TNF-α-treated HaCaTs-derived exosomes. (C) CD4 + T cells were transfected with LV-AGAP2-AS1 or LV-NC. The percentage of Th1 and Th17 cells was detected by flow cytometry. (D) The protein levels of IFN-γ and IL-17A are measured by ELISA. (E) Prediction of the binding sequence between AGAP2-AS1 and miR-424-5p and construction of the AGAP2-AS1 MUT sequence. (F) Dual-luciferase reporter gene assay confirmed the binding relationship between AGAP2-AS1 and miR-424-5p in HEK-293T cells. (G) AGAP2-AS1 expression level in CD4 + T cells transfected with miR-424-5p mimics or NC mimics and miR-424-5p inhibitor or NC inhibitor. WT wild type, MUT mutant type. Th, T helper. Experiments were repeated three times. *P < 0.05, **P < 0.01, ***P < 0.001. ns: no significance.

Article Snippet: For intracellular cytokine staining, cells were fixed with fixation buffer (E-CK-A109, Elabscience) and then stained intracellularly with antibodies against human IFN-γ (E-AB-F1196C, Elabscience) and IL-17A (E-AB-F1173D, Elabscience).

Techniques: Fluorescence, Microscopy, Labeling, Expressing, Incubation, Derivative Assay, Transfection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Binding Assay, Sequencing, Luciferase, Reporter Gene Assay, Mutagenesis

Journal: Frontiers in Genetics

Article Title: Exosomes containing long non-coding RNA AGAP2-AS1 promote the differentiation of CD4 + T cells through the miR-424-5p/SGK1 axis in psoriasis

doi: 10.3389/fgene.2025.1521470

Figure Lengend Snippet: AGAP2-AS1/miR-424-5p regulated CD4 + T cell differentiation via the SGK1 signaling pathway. (A) The relative expression levels of SGK1 in 293T cells that were transfected with siRNA targeting SGK1. (B) SGK1 expression level in CD4 + T cells transfected with LV-AGAP2-AS1 or LV-NC and si-AGAP2-AS1 or si-NC. (C) Western blot confirmed that transfection of AGAP2-AS1 in CD4 + T cells promoted the expression of SGK1, and the miR-424-5p mimics reversed this process. (D) Flow cytometry analysis of the percentages of Th1 and Th17 cells in CD4 + T cells transfected with LV-AGAP2-AS1 and miR-424-5p mimics or NC mimics. (E) The protein levels of IFN-γ and IL-17A are measured by ELISA. (F) Flow cytometry analysis of the percentages of Th1 and Th17 cells in CD4 + T cells transfected with LV-AGAP2-AS1 and SGK1 siRNA or NC siRNA. (G) The protein levels of IFN-γ and IL-17A are measured by ELISA. The experiments were repeated three times. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: For intracellular cytokine staining, cells were fixed with fixation buffer (E-CK-A109, Elabscience) and then stained intracellularly with antibodies against human IFN-γ (E-AB-F1196C, Elabscience) and IL-17A (E-AB-F1173D, Elabscience).

Techniques: Cell Differentiation, Expressing, Transfection, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Targeting Glutamine Metabolism Ameliorates Autoimmune Hepatitis via Inhibiting T Cell Activation and Differentiation

doi: 10.3389/fimmu.2022.880262

Figure Lengend Snippet: JHU083 inhibited T cells differentiation in vivo . Mice of Vehicle-AIH and JHU083-AIH group were intravenously injected with 8ml/kg ConA, and Vehicle-WT group was injected with normal saline of equal volume. JHU083-AIH group was gavaged with 0.3mg/kg JHU083 24h and 1h before ConA injection, and Vehicle-WT and Vehicle-AIH group were treated with equal vehicle at the same time points. (A) Flow cytometry was applied to analyze the Th1 cell percentage via IFNγ expression, the Th2 cell percentage via IL4 expression, and the Th17 cell percentage via IL17A expression. (B) ELISA was applied to analyze serum IFNγ (Th1 cells) and IL17 (Th17 cells) secretions. Flow cytometry was also applied to analyze the differentiation of CD8(+) T cells into cytotoxic T lymphocytes, evidenced by secreting IFNγ (C) and Granzyme (D) . Data were expressed as means ± SEM. * P < 0.05, ** P <0.01, and *** P <0.001. ns, not significant.

Article Snippet: The antibodies used in the flow cytometry were as follows: CD4 (FITC, E-AB-F1097C), CD8a (PE, E-AB-F1104D), CD25 (PE-Cy5, E-AB-F1102G), CD69 (PE-Cy7, E-AB-F1187H), IL4 (PE, E-AB-F1204UD), IL17A (APC, E-AB-F1272E) were purchased from Elabscience Biotechnology (Wuhan, China), antibodies IFNγ (APC-Cy7, cat# 561479) was purchased from BD Biosciences (UK), and Granzyme B (PE-Cy7, cat# 372213) was purchased from Biolegend (San Diego, CA, USA).

Techniques: In Vivo, Injection, Saline, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Targeting Glutamine Metabolism Ameliorates Autoimmune Hepatitis via Inhibiting T Cell Activation and Differentiation

doi: 10.3389/fimmu.2022.880262

Figure Lengend Snippet: DON treatment suppressed T cells differentiation in vitro . Freshly separated spleen cells were stimulated with 1.5mg/ml ConA with or without treating 2.5μM DON, and cells were analyzed 24h after ConA stimulation. (A) The differentiation markers of CD4+ T cells (IFNγ for Th1 cells, IL4 for Th2 cells and IL17 for Th17 cells) were detected using flowcytometry. (B) qRT-PCR was applied to analyze IFN-γ, IL4 and IL17 mRNA levels. (C) ELISA was applied to analyze supernatant IFN-γ and IL17 secretions. Flow cytometry was also applied to detect the production of activation markers of CTL, such as IFNγ (D) and Granzyme B (E) . Data were expressed as means ± SEM. ** P <0.01 and *** P <0.001, ns, not significant.

Article Snippet: The antibodies used in the flow cytometry were as follows: CD4 (FITC, E-AB-F1097C), CD8a (PE, E-AB-F1104D), CD25 (PE-Cy5, E-AB-F1102G), CD69 (PE-Cy7, E-AB-F1187H), IL4 (PE, E-AB-F1204UD), IL17A (APC, E-AB-F1272E) were purchased from Elabscience Biotechnology (Wuhan, China), antibodies IFNγ (APC-Cy7, cat# 561479) was purchased from BD Biosciences (UK), and Granzyme B (PE-Cy7, cat# 372213) was purchased from Biolegend (San Diego, CA, USA).

Techniques: In Vitro, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Activation Assay

Journal:

Article Title: High-Level Replication of Human Immunodeficiency Virus in Thymocytes Requires NF-?B Activation through Interaction with Thymic Epithelial Cells

doi:

Figure Lengend Snippet: TNF and, to a lesser extent, IL-1 are involved in the permanently induced NF-κB activity present in thymocytes during the coculture with TEC. Thymocytes were freshly isolated (lanes 1 and 2) or cultured for 45 h (lanes 3 to 11) either alone (lane 3 [Control thymocytes]) or in the presence of TEC under conditions avoiding contact (lanes 4 to 9) or allowing contact (lanes 10 and 11). During the coculture, the cells were either left untreated (lane 4 [Control coculture, 45 hours] and lane 10 [Control coculture]) or were treated at the start of the coculture with antibodies respectively raised against TNF (lane 5), IL-6 (lane 6), and IL-1β (lane 8), with the antagonist of the IL-1 receptor (lane 7), or with αTNF plus IL-1 ra plus αIL-6 (lanes 9 and 11). Whole-cell extracts from these various samples were incubated with a 32P-labeled oligonucleotide representing the HIV LTR-derived κB motif. Competition (comp [lane 1]) with a 40-fold molar excess of unlabeled oligonucleotide was used to confirm the specificity of the DNA-binding activity detected. NF-κB activity is indicated. The positions of two nonspecific (n.s 1 and n.s 2) binding activities are indicated. The positions of two nonspecific (n.s 1 and n.s 2) binding activities are indicated. This experiment is representative of three independent experiments, each carried out on a different thymus.

Article Snippet: MAb against IL-1β (B-A15) was purchased from Diaclone Research (Besançon, France).

Techniques: Activity Assay, Isolation, Cell Culture, Incubation, Labeling, Derivative Assay, Binding Assay

Journal:

Article Title: High-Level Replication of Human Immunodeficiency Virus in Thymocytes Requires NF-?B Activation through Interaction with Thymic Epithelial Cells

doi:

Figure Lengend Snippet: TNF and, to a lesser extent, IL-1 induce NF-κB activity in thymocytes, and IL-7 is required for this effect. Whole-cell extracts were prepared from freshly isolated thymocytes (panel A, lane 1) or from thymocytes cultivated for 30, 60, or 45 h. (A) During the indicated culture times, thymocytes were either left untreated (lane 1, control for freshly isolated; lane 2, control 30 h; and lane 5, control 60 h) or were stimulated with either TNF (lanes 3 and 6), IL-1β (lanes 4 and 7), or TNF in the presence of TEC CM (TEC CM + TNF) (lane 9). TEC conditioned medium was also added alone (lane 8 [TEC CM]). The data shown here are representative of three independent experiments carried out on three thymuses. (B) Thymocytes were cultured for 45 h either untreated (lane 1, control 45 h), with TNF (lane 2), with TEC CM (lane 3), or with TNF in the presence of TEC CM (TEC CM + TNF) preincubated (lane 5) or not (lane 4) with antibody against IL-7. In lanes 6 to 8, the thymocytes were left untreated for 6 h (lane 6) or incubated with an antibody against IL-7Rα (lane 7) or with an IgG1 control serum (lane 8) before being cocultured with TEC for 45 h. The data shown here are representative of two independent experiments carried out on two thymuses. (C) Cells were left untreated for the 45 h of the culture (lane 1, control 45 h) or treated with IL-1β (lane 2), TNF (lane 3), IL-1β plus TNF plus IL-6 plus GM-CSF (lane 4), IL-7 (lane 5), IL-7 plus IL-1β (lane 6), or IL-7 plus TNF (lane 7). This experiment is representative of three independent experiments carried out on three thymuses. (D) Thymocytes were left untreated (lane 1, control) or stimulated with IL-7 (lane 2) or IL-7 in presence of anti-TNF and Il-1ra (lane 3). Whole-cell extracts were incubated with a 32P-labeled oligonucleotide representing κB motif. NF-κB activity is indicated. This experiment is representative of two independent experiments, each carried out on a different thymus.

Article Snippet: MAb against IL-1β (B-A15) was purchased from Diaclone Research (Besançon, France).

Techniques: Activity Assay, Isolation, Cell Culture, Incubation, Labeling

Journal: The Journal of Experimental Medicine

Article Title: The Runx1 Transcription Factor Inhibits the Differentiation of Naive CD4 + T Cells into the Th2 Lineage by Repressing GATA3 Expression

doi: 10.1084/jem.20021200

Figure Lengend Snippet: Effect of cotransduction of GATA3 and Runx1 on Th2 cell differentiation. (A) Naive CD4 + T cells were isolated from the spleens of wild-type and GATA3 -transgenic mice, TCR-stimulated, infected by retroviruses carrying pMX or pMX-Runx1, and cultured in the presence of IL-2. The GFP + population was sorted and reactivated via TCR. The cytokines secreted into the culture supernatant were measured by ELISA. The values obtained for the pMX-Runx1–infected cells are presented as percent inhibition of secretion, taking the values observed for the pMX-infected cells to be 100%. (B) Naive CD4 + T cells were isolated from the spleens of IL-4R (−/−) mice, TCR-stimulated, coinfected by pMX-GFP and pMX-rat CD2 retroviruses as indicated, and cultured in the presence of IL-2. The rat CD2 + population was sorted, TCR-stimulated, and processed for flow cytometrical analysis of intracellular IL-4. Data are representative of two independent experiments.

Article Snippet: Anti-rat CD2 PE (LFA-2) was purchased from Cedarlane.

Techniques: Cell Differentiation, Isolation, Transgenic Assay, Infection, Cell Culture, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: Cell reports

Article Title: TLR9 Sensing of Self-DNA Controls Cell-Mediated Immunity to Listeria Infection via Rapid Conversion of Conventional CD4 + T Cells to T reg

doi: 10.1016/j.celrep.2020.01.040

Figure Lengend Snippet: (A and B) Tlr2 , Tlr4 , Tlr7 , and Tlr9 mRNA normalized to Hprt1 mRNA in (A) whole spleen and (B) sorted DC and T cell populations in C57BL/6 mice infected with LD or HD Lm Δ actA -Ova (n = 3 per group). (C and D) p65-GFP knockin mice challenged for 24 h with HD Lm Δ actA -Ova. (C) Frequency of p65-GFP and TLR9 expression in B cells, CD8a − DC/Mac, CD8a + DCs, plasmacytoid DCs (pDCs), CD103 + DCs, red pulp macrophages (RP Macs), and marginal zone Macs (MZ Macs) (n = 3 per group). (D) Representative ImageStream images displaying TLR9 hi APC subcellular localization of p65-GFP (green) and DAPI (red) and quantitative p65-GFP nuclear translocation (n = 3 per group). (E) C57BL/6 and Tlr9 M7Btlr mice exposed to HD Lm Δ actA -Ova for 24 h. Frequency and absolute number of IL-12 (p40) + and IFN-β + APC subsets after PMA/ionomycin stimulation (n = 4 per group). (F) C57BL/6 and Batf3 −/− mice infected with HD Lm Δ actA -Ova for 24 h. Change in FoxP3 + :FoxP3 − CD4 + T cell ratio at day 1 post-infection relative to the naive state (D FoxP3 + :FoxP3 − ) (n = 7 per group).

Article Snippet: ant-IL-12 (p40) (C17.8) PE , Tonbo Biosciences , Cat#50-7123-U100.

Techniques: Infection, Knock-In, Expressing, Translocation Assay

Journal: Cell reports

Article Title: TLR9 Sensing of Self-DNA Controls Cell-Mediated Immunity to Listeria Infection via Rapid Conversion of Conventional CD4 + T Cells to T reg

doi: 10.1016/j.celrep.2020.01.040

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ant-IL-12 (p40) (C17.8) PE , Tonbo Biosciences , Cat#50-7123-U100.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Staining, Gene Expression, Software, Luminex